Oestradiol Stimulates Morphological and Functional Differentiation of Human Villous Cytotrophoblast

Trophoblast differentiation is a complex process involving interactions of cytotrophoblastic cells with their evolutive milieu. During pregnancy, the feto-placental unit produces large amounts of steroids. Progesterone and oestradiol are increasingly produced when the syncytiotrophoblast is highly differentiated. Furthermore, receptors to these hormones are expressed by the trophoblast. This led us to test the hypothesis that steroid production could affect the morphological and functional differentiation of the trophoblast during gestation.The fusion of cytotrophoblastic cells into syncytiotrophoblast was assessed using fluorescence recovery after photobleaching for gap junctional communication analysis (gap-FRAP), desmoplakin immunostaining and connexin 43 expression. In parallel, functional differentiation was assessed by beta-human chorionic gonadotrophin (betahCG) production and human chorionic somatomammotropin (hCS) expression analysis. The presence of oestradiol, 1 microm, increased the percentage of coupled cells (3. 8-fold), connexin 43 expression and stimulated the syncytium formation. In parallel, oestradiol (1, 3 and 5 microm) induced a significant increase in the daily hCG production. The steroid action was specific, as the stimulatory effects were inhibited by tamoxifen. Oestradiol also stimulated hCS expression (51 per cent compared to control after 3 days). As trophoblastic differentiation is specifically stimulated by hCG, oestradiol could act via the stimulation of hCG production or via a direct action. In the presence of an efficient concentration of hCG antibody, oestradiol still stimulated hCS expression, suggesting a self-sufficient effect of the steroid. Physiological concentrations of progesterone were ineffective in modulating trophoblast differentiation.In conclusion, oestradiol could be implicated in the maturation and aging of the trophoblast.     

Adhesion Molecules in Human Trophoblast – A Review. II. Extravillous Trophoblast

At the tips of anchoring villi, cytotrophoblast (CTB) proliferation leads to a process of multilayering in which cells lose their attachment to the villous basement membrane and develop into columns, within which they adhere to one another using desmosomes, with associated intermediate filament bundles. Non-desmosomal cadherins, tight junction proteins and other adhesion molecules are also present, suggesting that actin-associated adhesions contribute to placental anchorage. In the distal columns, cell–cell interactions diminish, cells upregulate β1 integrins and bind to a provisional fibrinoid extracellular matrix, eventually detaching to migrate into the decidual stroma and myometrium, where interstitial and endovascular extravillous trophoblast (EVT) populations show distinct repertoires of adhesion molecules.     

Localization of Angiogenic Growth Factors and Their Receptors in the Human Placental Bed Throughout Normal Human Pregnancy

During early human pregnancy invasion of uterine spiral arteries by extravillous trophoblast cells contributes to their remodelling characterised by loss of musculo-elastic media and replacement by fibrinoid containing trophoblast. Despite its importance for successful pregnancy, the mechanisms underlying ‘transformation’ of spiral arteries are not well understood. The aim of this study was to localize expression of members of the angiopoietin (Ang) family (Ang-1, Ang-2 and their receptor Tie-2) and the vascular endothelial growth factor (VEGF) family (VEGF-A, VEGF-C, VEGF-D and their receptors VEGF-R1, VEGF-R2 and VEGF-R3) in the placental bed throughout normal human pregnancy. Placental bed biopsies were obtained from women undergoing elective termination of pregnancy at 8–10, 12–14 and 16–20 weeks' gestation and elective caesarean section at term (n = 6 each group). Paraffin-embedded sections were immunostained for Ang-1, Ang-2, Tie-2, VEGF-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2 and VEGF-R3 using an avidin biotin peroxidase technique. Reactivity of endovascular, interstitial, intramural and multinucleate extravillous trophoblast populations in the placental bed was analysed semi-quantitatively. There was an increase in the level of immunostaining of intramural EVT for Tie-2 and VEGF-C with increasing gestational age. In addition, there was a reduction in Ang-1 and Ang-2 expression by multinucleate interstitial EVT and of VEGF-R1 and VEGF-R2 by endovascular EVT with increasing gestational age. At the earlier gestational ages studied, immunostaining for Ang-1, Ang-2, Tie-2, VEGF-C, VEGF-R1 and VEGF-R2 on intramural EVT was reduced compared to both mononuclear interstitial and endovascular EVT. These findings suggest that the Ang and VEGF families may play a role in the process of spiral artery remodelling in normal pregnancy.     

甲氧滴滴涕对人绒毛外滋养细胞侵袭能力的影响

目的:探讨甲氧滴滴涕(MXC)对绒毛外滋养细胞侵袭能力的影响。方法:用胰蛋白酶消化后再经流式细胞仪分离得到人绒毛外滋养细胞进行原代培养及鉴定。细胞分为MXC(1μmol/L,3μmol/L、5μmol/L、7μmol/L)处理组及空白对照组,培养12h、24h和48h。用侵袭小室检测每组各时间点的侵袭能力,Western blot检测各组各时间点MMP-2、MMP-9、TIMP-2、TIMP-1表达,RT-PCR法检测MMP-9 mRNA、MMP-2 mRNA表达。结果:MXC处理组滋养细胞侵袭能力呈时间剂量依赖型下降,MMP-9 mRNA、MMP-2 mRNA表达下降,MMP-9、MMP-2表达下降,TIMP-2、TIMP-1表达增高。MMP-2及其mRNA下降程度与MXC有时间剂量效应。结论:MXC可通过改变基质金属蛋白酶及其抑制物的比例降低绒毛外滋养细胞侵袭能力。     

Adhesion Molecules in Human Trophoblast – A Review. I. Villous Trophoblast

In the placental villus, cells attach to basement membrane via integrin α6β4 and adhere both laterally and apically to their neighbours. The most prominent adhesive specialisation seen using the electron microscope is the desmosome, which connects cytotrophoblast cells (CTB) laterally and also contributes to the attachment of CTB to the overlying syncytium. However, numerous cadherins and other junctional proteins are also present in the corresponding plasma membrane domains, indicating a multiplicity of adhesive interactions. Integrins, tight junction components and cadherins are all found in the syncytial microvillous membrane, perhaps reflecting its ability to form intersyncytial bridges. There is a wide gulf to be filled between molecular anatomy and functional studies, with much to be learned about the role of adhesion molecules in regulating villous epithelial integrity, homeostasis and growth.     

人早孕期蜕膜和绒毛外滋养层细胞的免疫组织化学研究

侵入蜕膜组织的绒毛外滋养层细胞在HE染色标本上不易辨认。我们用免疫组织化学,ABC法,过氧化物-抗过氧化物酶标记蜕膜组织中的hCG与hPL阳性细胞。结果证明,子宫内膜中只要有一个绒毛外滋养层细胞存在就能被显示出来。这对提高妊娠诊断,防止残留滋养层细胞的增生与恶性病变很有意义。我们在蜕膜组织中还发现3类胎盘激素阳性细胞:hCG阳性细胞,hPL阳性细胞及hCG与hPL均为阳性的细胞。这一发现,证明了蜕膜有内分泌功能。     

米非司酮对早孕绒毛组织Notch/Snail/E-钙黏素表达的影响

目的:初步探讨米非司酮抗早孕的作用机制和Notch信号转导通路的关系。方法:采用实时荧光定量逆转录聚合酶链反应技术(TR-qPCR)和免疫组织化学技术检测正常早孕绒毛组织中及不同剂量米非司酮作用后绒毛组织中Notch信号转导通路相关分子Notch-1、Snail、E-钙黏素(E-cadherin)在mRNA水平及蛋白水平的表达情况。结果:米非司酮对早孕绒毛组织的作用以200mg组(D组)最为显著,Notch-1、Snail基因以及E-钙黏素蛋白表达较对照组(A组)、100mg组(B组)、150mg组(C组)作用明显,差异有统计学意义(P<0.05);B组与A组比较差异无统计学意义(P>0.05)。C组Notch-1mRNA、Snail蛋白与A组比较差异有统计学意义(P<0.05),其余各基因及蛋白表达差异均无统计学意义(P>0.05)。结论:米非司酮抗早孕机制可能和Notch信号转导通路有关。     

绒毛外滋养细胞与螺旋动脉重铸及子痫前期

妊娠期高血压疾病是妊娠期特有疾病,迄今仍是孕产妇和围生儿死亡的主要病因之一.子痫前期是该病的常见和主要的发病类型,其病因和发病机制至今尚未完全阐明.国外学者利用活体妊娠动物模型深入研究,从分子细胞层面揭示了螺旋动脉重铸的过程,主要包括绒毛外滋养细胞的侵入及其介导的血管平滑肌细胞和内皮细胞的凋亡,提出许多影响这一过程的因...     

人蜕膜和绒毛膜组织在鸡胚绒毛尿囊膜的种植和在体培养观察

来源于人流组织的蜕膜和绒毛膜,异种接种于发育8d的鸡胚绒毛尿囊膜(ChickenChorioallantoic Membrane,CAM)上,进行在体培养观察10d.将378块绒毛膜和808块蜕膜组织分别种植于69和122个鸡胚,鸡胚胎存活率分别为86.96%和98.36%(P<0.01),种植组织成活率分别为93.62%和93.81%;种植组织于在体培养过程中,CAM表面产生血管增生及出血现象,出血率分别为41.67%和20.37%(P<0.05).两种组织种植后均无异种排斥反应和炎症反应发生.建立CAM在体培养模型,简便快速,观察方便,可同时进行大样本实验,是一种很好的实验手段.     

孕酮对早孕人滋养层HLA-G mRNA转录水平的调控

目的:探讨孕酮对人早孕滋养层人类白细胞主要组织相容性抗原(HLA)-G基因转录的调控作用。方法:选取正常妊娠6-9周的人绒毛标本,进行组织块贴壁培养、纯化获得滋养层细胞后,采用半定量逆转录-聚合酶链式反应(RT-PCR)检测孕酮对体外培养的人滋养层细胞HLA-G基因转录的影响。结果:添加孕酮的滋养层细胞HLA-GmRNA水平显著增高(P<0.05)。结论:孕酮对滋养层HLA-G的转录具有明显的上调作用。     

人早孕期绒毛和绒毛外细胞滋养细胞的分离、培养及鉴定

目的:建立人早孕期绒毛细胞滋养细胞(villous cytotrophoblasts,VCT)及绒毛外细胞滋养细胞(extravillous cytotrophoblasts,EVCT)的分离、培养方法。 方法:利用不同的胰酶消化条件,收集人早孕期绒毛组织的VCT及EVCT并分别培养。倒置显微镜、扫描电镜观察VCT及EVCT的形态学特征;免疫细胞化学鉴定细胞来源及纯度。 结果:胰酶短期、温和消化获得的EVCT,可生长于Matrigel包被的培养器皿上,并表达特异性标志物c-crbB-2;延长消化时间、增加胰酶浓度获得的VCT,种植于塑料或玻璃培养器皿上可聚集并融合形成合体滋养细胞。VCT不表达c-crbB-2。结论:采用不同的胰酶消化及体外培养条件,可简单、快捷地分别获得高纯度人早孕期VCT及EVCT。     

胰岛素样生长因子-Ⅰ对体外人滋养层细胞孕酮分泌的影响

目的:探讨胰岛素样生长因子-I(IGF-I)对人早孕绒毛滋养层细胞孕酮(P)的合成和调节 作用。方法:将胰蛋白酶和胶原酶联合消化人早孕绒毛滋养组织,Percoll密度梯度分离纯化后得 到的人早孕胎盘滋养层细胞进行原代培养。以终浓度为0.1μg/L、1μg/L、10μg/L、100μg/L　IGF-I分 别对其作用12h,以及100μg/L浓度IGF-I作用12h、24h、48h、72h时,放免法检测滋养细胞 分泌P 的含量,RT-PCR法检测低密度脂蛋白受体(LDLR)mRNA的表达。结果:滋养层细胞P 的 分泌量随着IGF-I的浓度升高而增加;同时100μg/L浓度的IGF-I作用于滋养层细胞12h 后,P 分泌开始增加,48h达到高峰,以后逐渐下降。半定量RT-PCR均显示LDLRmRNA阳性条带,且表 达规律与P一致。结论:滋养层细胞P的分泌具有对IGF-I的时间和浓度依赖性,并且IGF-I能 上调LDLRmRNA的表达,对促进滋养细胞P分泌的调节起重要作用。     

孕酮和IL-15对人早孕蜕膜子宫自然杀伤(uNK)细胞促血管生成因子的调节

目的:探讨孕酮和IL-15对蜕膜子宫自然杀伤细胞(uNK)在早孕蜕膜血管生成/重塑过程中的作用。方法:免疫磁珠(MACS)分离及纯化蜕膜uNK细胞后进行体外培养,分别用不同浓度孕酮或IL-15干预72h后获取培养上清液和细胞。采用RT-PCR和ELISA检测蜕膜uNK细胞的VEGF-A、VEGF-C、Ang2的mRNA转录和蛋白表达。结果:蜕膜uNK细胞可表达多种与血管形成有关的生物活性分子,与未干预的空白对照组相比,IL-15促进uNK细胞表达VEGF-A、VEGF-C(P<0.05),且呈剂量依赖关系,但对Ang2无影响;而孕酮对uNK细胞表达上述因子均无显著影响(P>0.05)。结论:妊娠早期蜕膜uNK细胞通过表达多种促血管生成因子,在早孕蜕膜血管生成/重塑中起着重要的作用,IL-15对此有直接促进作用。     

五味子乙素对BaP致HTR-8/SVneo侵袭力损伤的保护

目的：以人绒毛膜外滋养层细胞（human trophoblast HTR-8/SVneo,简称HTR）细胞株为研究载体,研究五味子乙素（Schisandrin B,Sch B）对苯并芘（benzo[a]pyrene,BaP）致HTR细胞侵袭力损伤的影响。方法：实验分为对照组、BaP染毒组、不同浓度Sch B（0.5,1,2 μmol/L）干预染毒组,通过新型四唑化合物（MTS）法检测细胞增殖情况,明确BaP染毒及Sch B干预剂量;通过Transwell侵袭实验检测BaP染毒后以及Sch B干预后细胞侵袭力的改变。结果：①MTS检测细胞增殖情况：与对照组相比,20 μmol/L浓度BaP作用后细胞增殖明显下降（P＜0.01）,不同浓度Sch B（0.5,1,2 μmol/L）干预后细胞增殖情况优于BaP染毒组（P＜0.05）。②Transwell细胞侵袭实验：与对照组相比,BaP染毒组细胞侵袭力明显下降（P＜0.01）,不同浓度Sch B干预组细胞侵袭力也呈下降趋势（P＜0.05）;与BaP染毒组相比,Sch B干预组细胞侵袭力明显提升（P＜0.05）。结论：一定浓度的BaP会对HTR的细胞增殖及侵袭力造成损伤,Sch B可以预防BaP对HTR细胞增殖造成的损伤,同时提高细胞侵袭力。     

五味子乙素对BaP致HTR8-SVneo细胞损伤的保护作用及机制

目的：研究五味子乙素（Schisandrin B,Sch B）预防苯并芘（BaP）致人绒毛膜外滋养层细胞（HTR8-SVneo）损伤的作用机制。方法：通过新型四唑化合物（MTS）法观察和测定,最终选取0.5,1,2 μmol/L Sch B分别作用HTR8-SVneo细胞24 h后再给予20 μmol/L Bap染毒处理24 h,MTS检测各组细胞生长情况;JC-1荧光染色法观察各组线粒体膜电位荧光强度变化;酶联免疫吸附试验（ELISA）检测各组细胞内促凋亡蛋白（Bax）、细胞色素C（cytochromec,cyt-c）蛋白浓度;蛋白质印迹法（Western blotting）检测各组的凋亡蛋白（Caspase-9）。结果：①BaP染毒组OD值为1.187±0.015,0.5,1,2 μmol/L Sch B预保护24 h后的OD值分别为1.483±0.022、1.489±0.048和1.531±0.240,差异有统计学意义（P＜0.05）。②BaP染毒组线粒体荧光强度弱,0.5,1,2 μmol/L Sch B预保护24 h后的荧光强度较强,差异有统计学意义（P＜0.05）。③BaP染毒组Bax、cyt-c蛋白浓度升高,0.5,1,2 μmol/L Sch B预保护24 h后细胞内Bax、cyt-c蛋白浓度降低,差异有统计学意义（P＜0.05）。④BaP染毒组细胞内caspase-9蛋白表达升高,0.5,1,2 μmol/L Sch B预保护24 h后细胞内caspase-9蛋白表达降低,差异有统计学意义（P＜0.05）。结论：Sch B可以预防BaP致HTR8-SVneo细胞损伤,其作用机制可能与抗线粒体途径有关。